The present invention relates to the use of a combination of electric field energy and ultrasound energy for the selective ablation of tissues, such as tumor tissues, and preferably cells in an organism.
In general therapeutic applications of ultrasound in the clinic may be divided into two major categories; applications that employ low intensity (0.125-3 W/cm2) and those that employ higher intensities (xe2x89xa75 W/cm2) (ter Haar, 1999, Eur. J. Ultrasound 9: 3). Low intensity ultrasound is commonly used in applications such as physiotherapy, for example, to stimulate normal physiological responses to injury or to accelerate processes, such as the transport of drugs across the skin. Treatment with low intensity ultrasound rarely results in collateral tissue damage and usually extreme efforts are employed to minimise such effects. This includes minimising excessive tissue heating which results from exposure to ultrasound, typically by reducing the treatment time and/or delivering the ultrasound in a pulsed manner.
In contrast, the major objective of applications involving the use of high intensity ultrasound is to selectively destroy tissue by hyperthermic processes. High intensity ultrasound-mediated tissue ablation may be categorised according to the way in which ultrasound energy is delivered to tissues. Ultrasound may be delivered directly from a transducer to the treatment area; alternatively, a coupling device which focuses the ultrasound may mediate delivery. When provided through a coupling device, ultrasound passing through intervening tissues is usually low intensity and therefore, relatively non-destructive. However, at the focal point, the energy which has accumulated is raised to a pre-determined higher intensity and tissue destruction occurs at, or around, the focal point. Thus, using a coupling device has the advantage of selectively delivering ablative energy to a tissue being treated without causing major damage to intervening tissues.
In general, therapeutic applications which rely on the use of high intensity focussed ultrasound, or xe2x80x9cHIFU,xe2x80x9d exploit the heat which is generated at the focal point and a number of methods together with devices for achieving focus and tissue ablation have been suggested (see, e.g., U.S. Pat. Nos. 4,888,746; 5,895,356; 5,938,608 and International Patent Applications WO 97/35518 A1 and WO 99/22652 A1).
In addition to a requirement for relatively sophisticated equipment to achieve focussing of high intensity ultrasound, one major disadvantage associated with the use of HIFU involves the potential for the occurrence of cavitation events which, in turn, leads to the formation of destructive, or possibly mutagenic, free radicals (Miller et al., (1996) Ultrasound in Med. and Biol. 22: 1131). An alternative approach involving a mechanism of sensitising the target tissue to low intensity ultrasound (either focussed or non-focussed) would therefore provide an advantage.
It has been found that the delivery of short, intense electric pulses to cell populations or tissues in vivo results in transient permeabilisation of cell membranes and this has provided the basis for what has become known as electrochemotherapy (Heller et al., (1999) Advanced Drug Delivery Rev. 35: 119). Electrochemotherapy was originally developed to facilitate the passage of chemotherapeutic drugs into cancer cells which had become impermeable to those drugs. The technique has developed to a stage where delivery of electric pulses in vivo is being exploited in areas such as gene therapy in order to mediate introduction of DNA to target tissues. Devices designed to facilitate delivery of electric pulses in vivo under a variety of conditions (e.g., transdermal, laparoscopic, catheter-mediated delivery, etc.) currently exist (see, e.g., International Patent Applications WO 99/22809 A1, WO 99/06101 A1; WO 99/01157 A1, WO 99/01157 A1, and WO 99/01158).
More recently, it has been found that exposure of human erythrocytes to short and intense electric pulses which facilitates transient permeabilisation also results in a dramatic sensitisation to low intensity ultrasound (WO 01/07011).
The present invention relies partially on the discovery that sensitisation of nucleated cells by application of an electric field (xe2x80x9celectrosensitisationxe2x80x9d) renders the cells susceptible to ablation using low intensity ultrasound and thereby provides a means of eliminating unwanted cells and tissues in the body. The invention also relies on the discovery that exposure of a cell to ultrasound followed by exposure to electric fields also results in cell disruption. Thus, exposure of a nucleated cell to ultrasound and an electric field applied in any order, results in cell disruption.
In one aspect, the invention provides a method of rendering a cell sensitive to disruption by ultrasound comprising exposing the cell to an electric field for an amount and time sufficient to render it more susceptible to disruption by ultrasound than an unsensitised cell. The invention also provides a method of disrupting a cell comprising providing a cell which has been exposed to an electric field for an amount and time sufficient to render it more susceptible to disruption by ultrasound than an unsensitised cell; and exposing the cell to ultrasound, thereby disrupting the cell. The invention further provides a method of disrupting a cell comprising exposing the cell to an electric field for an amount and time sufficient to render it more susceptible to disruption by ultrasound than an unsensitised cell; and exposing said cell to ultrasound, thereby disrupting the cell. The cell can be part of a tissue, such as a tumor tissue, and exposure to ultrasound can be performed in vitro, ex vivo, or in vivo. Similarly, the step of exposing the cell to the electric field can occur in vitro, ex vivo, or in vivo. Disruption can be a result of apoptosis of the cell and in a preferred aspect, disruption results in ablation of cells from the body of an organism.
In one aspect, the method further comprises the step of exposing the cell to an agent which facilitates cell death. The cell-death facilitating agent can be selected from the group consisting of an oligonucleotide, a ribozyme, an antibody, an enzyme, a cytotoxic agent, a cytostatic agent, a cytokine, GM-CSF, IL-2, an immunogen, and combinations thereof.
In one aspect, the electric field to which the cell is exposed is from about 1 Volt/cm to 10 kVolts/cm. The electric field is preferably applied for between about 1 xcexcs and 100 milliseconds. Ultrasound is preferably applied at a power level of from about 0.05 W/cm2 to 100 W/cm2 and continuous wave ultrasound or pulsed wave ultrasound can be applied.
In one aspect, the invention provides a method for disrupting a cell which comprises exposing the cell to a sensitising stimulus. The sensitising stimulus makes a cell more susceptible to a disrupting stimulus than a non-sensitised cell. The cell is then exposed the cell to a disrupting stimulus under conditions suited to disrupt the sensitised cell while not substantially disrupting non-sensitised cell.
In another aspect, the invention provides a method for selectively disrupting one or more target cells at a target site comprising a plurality of cells comprising: (a) exposing the one or more cells to an electric field; and (b) exposing the one or more cells to ultrasound; wherein the exposing in steps (a) and (b) disrupts the one or more target cells.
Preferably, the electric field is a low intensity electric field which has an electric field strength of less than 20 V/cm. In one aspect, direct current is applied to expose the one or more cells to the low intensity electric field. In another aspect, the current is between 100 xcexcA to 200 mA.
In one aspect, the method is performed using an electric field is from 1 Volt/cm to 10 kVolts/cm under in vivo conditions. In another aspect, the electric field is applied for between 1 xcexcs and 100 milliseconds. In a further aspect, ultrasound is applied at a power level of from 0.05 W/cm2 to 100 W/cm2. Ultrasound can be continuous wave ultrasound and pulsed wave ultrasound.
In one aspect, the one or more target cells comprise abnormally proliferating cells. For example, the one or more target cells can comprise tumor cells from a tumor which may be benign or malignant.
In another aspect, the one or more target cells are skin cells. The target site can comprise a wart, a papiloma, a psoriatic region of skin, a region of skin with eczema, or a mole.
In another aspect, the target site comprises a plurality of cells in a fluid, for example a buffer, cell culture medium or a bodily fluid. For example, the method can be performed at least partially in vitro, such as in a container or an extracorporeal circulating device. In another aspect, target site is within the body of an organism and the method is performed in situ. The target site can be exposed through an open surgical field or can be accessed using a medical access device which brings a source of electrical energy and ultrasound energy in sufficient proximity to the target site, to disrupt said one or more target cells.
The medical access device can be selected from the group consisting of a catheter, an endoscope, and a laparoscope.
The method can be used to remove unwanted cells for either therapeutic or cosmetic purposes. In one aspect, the target site comprises an unwanted fatty deposit, such as a lipoma and the one or more target cells comprise adipose cells.
Exposing in steps (a) and (b) can be performed over repeated intervals; for example, to remove a cell growth that may reoccur (e.g., such as a cancer).
Exposing can also be used to debulk tissue. In one aspect, the one or more cells are at the site of a wound. In another aspect, the one or more cells are within a blood vessel. In a further aspect, the target site comprises benign granulomatous tissue.
In one aspect, step (a) of the method comprises providing one or more pulses of electrical energy; preferably, multiple pulses are provided.
In another aspect, step (b) comprises providing low intensity ultrasound.
In one aspect, exposing in step (a) sensitises the one or more target cells and exposing in step (b) disrupts the one or more target cells. However, in another aspect, exposing in step (b) sensitises the one or more target cells and exposing in (a) disrupts the one or more target cells, and step (b) is performed prior to step (a).
In one aspect, exposing in steps (a) and (b) induces apoptosis in the one or more cells. Disrupting preferably results in cell death and/or cell ablation. Preferably, cells disrupted in the body of an organism are removed from the body of the organism. For example, removal of cells can be effected by immune response cells. In one aspect, therefore, the method comprises administering to the organism an agent which modulates an immune response.
In a further aspect, the method comprises the step of exposing the one or more target cells to an agent for facilitating cell death. The cell-death facilitating agent can be selected from the group consisting of an oligonucleotide, a ribozyme, an antibody, and enzyme, a cytotoxic agent, a cytostatic agent, a cytokine, GM-CSF, IL-2, an immunogen, and combinations thereof.
The invention also provides a system for disrupting a cell. The system comprises an electrosensitisation module comprising an electrical field generator for sensitising a cell to render it susceptible to disruption by an energy source, thereby to produce a sensitised cell, and an ultrasound generating module in communication with the electrosensitisation module for delivering ultrasound energy to said sensitised cell at a level sufficient to disrupt said cell. Either module can be provided separably, or as integrated but removable units, or as integrated but non-removable units. When the modules are part of an integrated system, they can be placed in fluid communication with each other such that cells can move from one module to another.
Preferably, the electric field generator delivers an electric field at a sufficient energy level and for a sufficient amount of time such that the sensitised cell is rendered more susceptible to disruption by ultrasound than an unsensitised cell. Preferably, the electric field generator can generate an electric field from 1 Volt/cm to 10 kVolts/cm in vivo. In one aspect, the ultrasound generating module is capable of generating ultrasound at a power level of from 0.05 W/cm2 to 100 W/cm2.
In another aspect, the electrosensitisation module comprises a housing having a lumen and the ultrasound module is positioned within the lumen. The ultrasound module can be removable from said electrosensitisation module. In a further aspect, the system comprises an insulation ring comprising a material which absorbs ultrasound and which insulates the electric field generator from short circuit discharge.
Preferably, the electric field generator comprises one or more electrodes. In one aspect, the electrodes are in the form of an array of needles. In another aspect, the electrosensitisation module comprises a delivery element comprising a housing defining a lumen through which fluids (e.g., cell-death facilitating agents, therapeutic agents, and the like) can be delivered to a target site.
Preferably, the system is in communication with a power source. More preferably, the system is in communication with a processor which can be programmed to implement operating parameters for the system.
The invention also provides a medical access device comprising a housing defining a lumen and wherein the system described above is positioned within the lumen. Preferably, the system is removable from the medical access device. The medical access device can comprise one or more of an irrigation source, irrigation channels, an optical system for transmitting light to and receiving light from a target site, or a cutting element. In one aspect, the medical access device is selected from the group consisting of a catheter, an endoscope, and a laparoscope.
The invention further provides an electrosensitisation module comprising a housing defining a lumen and an electric field generator in communication with the lumen which is suitable for generating an electric field within the lumen which is not of sufficient strength to electroporate a cell placed within the lumen, but is of sufficient strength to sensitise a cell so that it is more sensitive to disruption than a non-sensitised cell.
The invention also provides a method of inducing apoptosis in a cell. The method comprises exposing the cell to an electric field and exposing the cell to ultrasound.
In another aspect, the invention provides a method of identifying a gene product which is involved in an apoptotic process. The method comprises the steps of: inducing apoptosis in a cell by exposing the cell to an electric field and ultrasound and detecting a gene product whose expression changes in response to the exposing. The expression of the gene product can be induced, up-regulated, or down-regulated. In a further aspect, the method comprises the steps of altering the expression of a gene product or a gene encoding the gene product in a cell, exposing the cell to an electric field, exposing the cell to ultrasound, and measuring apoptosis. Apoptosis can be measured by detecting the presence or absence of fragmented DNA in a sample of nucleic acids from the cell. Apoptosis also can be measured by observing morphological features of the cell, by measuring the amount of histone complexes comprising fragmented DNA in a sample, by measuring the activity or determining the presence of proteases characteristically involved in apoptotic processes (e.g., such as caspase 3), or by other assays routinely performed in the art.
In one aspect, the invention provides a method of identifying a molecule which affects apoptosis of a cell. The method comprises the steps of: contacting a cell with a candidate molecule, exposing the cell to an electric field, exposing the cell to ultrasound and determining whether apoptosis is affected as a result of the contacting. In one aspect, the step of determining comprises measuring the expression of one or more genes involved in an apoptotic pathway. This measuring can comprise measuring one or more of: the amount, localisation, or function of gene product(s) of the one or more genes. In one embodiment, the step of determining comprises detecting the fragmentation of DNA in the cell.
The invention also provides a gene or gene product or a molecule identified according to the methods described above.